Complete Genome Sequences of BK Polyomavirus Strains from Two Patients with Urinary Tract Infection, Sequenced Using the Ion Torrent Platform.
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ABSTRACT:
SUBMITTER: Rose G
PROVIDER: S-EPMC5690343 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time (T90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated (T90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 (T90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid.IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate.
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Project description:Polyomavirus BK (BKV) has emerged as an important pathogen in kidney transplant patients. Existing taxonomic classifications of BKV come from conventional DNA sequence alignments based on limited data derived from the VP1 gene. We have used a phylogenetic whole-genome approach to examine the pattern of diversity and evolutionary relationships between 45 BKV strains isolated from multiple clinical settings. This analysis supports the classification of BKV into six genotypes, of which types V and VI have not been previously recognized. BKV strains hitherto classified as type I are, in fact, quite heterogeneous, and several cluster with our newly defined genotypes V and VI. The sequence information needed for assigning genotypes can be captured by VP1, VP2, VP3, or large T-gene sequencing. The most polymorphic coding region in the viral genome is VP1, but significant variation is also present in the large T-antigen gene, wherein polymorphisms are found in 11.39% of all nucleotide sites, 46.22% of which are cluster specific. Type-specific amino acid changes within the VP1 region are predicted to map to the BC and DE loops. The number of taxonomically informative amino acid changes in the large T antigen exceeds even that of the VP1 region. Viral strains isolated from healthy subjects and from patients with human immunodeficiency virus infection, Wiskott-Aldrich syndrome, and vasculopathy with capillary leak syndrome formed distinct subclusters. However, within the kidney transplant population, BKV strains derived from patients with asymptomatic viruria did not show complete separation from strains associated with allograft nephropathy.
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Project description:Two polyomaviruses, BK virus (BKV) and JC virus (JCV), are ubiquitous in the human population, generally infecting children asymptomatically and then persisting in renal tissue. It is generally thought that reactivation leads to productive infection for both viruses, with progeny shed in the urine. Several studies have shown that the rate of JC viruria increases with the age of the host, but a systematic approach to examine the shedding of BKV has not been developed. To elucidate the relationship between BK viruria and host age, we obtained urine from donors (healthy volunteers or nonimmunocompromised patients) who were divided into nine age groups, each containing 50 members. A high-sensitivity PCR was used to detect BKV and JCV DNA from urinary samples, and the specificity of amplification was confirmed by sequencing or restriction analysis of the amplified fragments. The rate of BK viruria was relatively low in subjects aged <30 years but gradually increased with age in subjects aged > or =30 years. However, BK viruria was less frequent than JC viruria in adults. The detected BKV isolates were classified into subtypes, and detection rates for individual subtypes were compared among age groups; this analysis showed that viruria of subtypes I (the most prevalent subtype) and IV (the second most prevalent subtype) occurred more frequently in older subjects. Therefore, our results reveal new aspects of BK viruria in nonimmunocompromised individuals.
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Project description:5' selective RNA-seq of 47 Single HEK293 cells RNAseq profiling with N4H4 unique molecular identifiers processed on a Fluidigm C1.
2016-12-19 | GSE79130 | GEO
Project description:We show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic cells is comparable to those obtained by the conventional microarray-based method.
2018-10-19 | GSE115847 | GEO
Project description:Data on polyomavirus genomic diversity has greatly expanded in the past few years. The implications of viral DNA sequence variation on the performance of molecular diagnostic assays have not been systematically examined. 716 BK, 1626 JC, and 73 SV40 virus sequences available in GenBank were aligned using Clustal-X. Five different published BKV PCR assays currently in use at major medical centers were evaluated for primer and probe mismatches with available GenBank sequences. Coverage of naturally occurring BKV strains varied amongst different assay methods. Targeted viral sequences showed major mismatch with primer or probe sequence in up to 30.7% of known BKV strains. BKV subtypes IVa, IVb, and IVc were more prone to this problem, reflecting common use of Type I Dun sequence for assay design. Despite the known polymorphism of this gene, 484 VP-1 sequences with conserved areas potentially suitable for PCR assay design are available. Assay targets in the Large T-antigen and agnogene are less subject to genetic variation, but sequence information corresponding to the latter two genes is available only for 164 and 174 published strains, respectively. Cross reactivity of appropriately selected BKV primers with JCV and SV40 sequences available in current databases was not a significant problem.
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Project description:Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.
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Project description:BK virus (BKV) is a polyomavirus that cause of allograft dysfunction among kidney transplant recipients. The role of BKV infection in non-renal solid organ transplant recipients is not well understood neither for the relationship between various BKV strains with occurrence of BKV viral viruria. This study aimed to understand the prevalence of BKV infection and identified of BKV various strains in the urine of liver transplant recipients. There was not significant difference of renal outcome between high BKV viruria and low BKV viruria in the liver transplant recipients. The WW-non-coding control region (NCCR) BKV detected in urine was associated with higher urinary BKV load, whereas the Dunlop-NCCR BKV was detected in the urine of low urinary BKV load. An in vitro cultivation system demonstrated that WW-BKV strain exhibiting the higher viral DNA replication efficiency and higher BKV load. Altogether, this is the first study to demonstrate the impact of BKV strains on the occurrence of BK viruria in the liver transplant recipients.
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Project description:Hepatitis B virus (HBV) is among the most common causes of liver cirrhosis. We report the full-genome sequences of seven molecular clones of HBV genotype A, amplified from an HBV-infected North Indian patient. This is probably the first report of the HBV genome sequencing using Ion Torrent from India.
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Project description:A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
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